Chemotaxis plays an essential role in immunity, angiogenesis, wound healing, and neuronal guidance and studies of its basic mechanisms are expected to present new approaches in the treatment of inflammation, metastasis and cancer progression, asthma, and arthritis. Many chemoattractants along with hormones, neurotransmitters, odorants and other sensory stimuli exert their effects upon cells through a vast family of serpentine G-protein couple receptors (GPCRs). The proposed studies focus on chemoattractant-mediated activation of G-proteins and the connection of these signaling events to directed cell migration. Studies in the previous grant period led to the development of a fluorescence resonance energy transfer (FRET)-based sensor that directly reports of the state of association Galpha- and Gbeta-gamma-subunits in living cells. This sensor will be applied to determine the spatial pattern of G-protein activation in D. discoideum cells oriented in chemoattractant gradients and to determine the effects of alpha- and beta-subunit point mutations, RGS proteins, and receptor phosphorylation on the rate constants of the heterotrimeric G-protein cycle in vivo. The technique will be combined with single molecule imaging to observe the transient activation of individual G-proteins during unitary receptor occupancy events. FRET sensors for members of the mammalian G-protein families Gs, Gi, Gq, and G12 will be built and similar experiments carried out in mammalian tissue culture cells. A combination of genetic and biochemical approaches are planned to discover the connections of G-protein signaling to directed motility. First, novel PH-domain containing proteins will be localized to the leading edge of chemotaxing cells and disrupted to determine their roles in chemotaxis. Second, genes involved in gradient sensing will be identified by suppression of known signaling mutants and in visual screens for defects in PH-GFP translocation. Third, the roles of YakA, TsuA, and TorA, identified in the last grant period as specifically required for sensing or movement, will be elucidated.